Precision Editing of Myosin Phosphatase for Vasodilator Sensitization in Hypertension

肌球蛋白磷酸酶的精确编辑对高血压血管舒张剂的敏感性

基本信息

批准号：
10338049
负责人：
Steven A. Fisher
金额：
$ 34.76万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-20 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10338049
关键词：
Address Adolescence Adolescent Aftercare Alternative Splicing Angiotensin II Animal Model Animals Arteries Biological Assay Biological Availability Birth Blood Pressure Blood Vessels Blood flow C-terminal CRISPR/Cas technology Cardiovascular system Cause of Death Code Cre-LoxP Cyclic GMP Development Dilator Disease susceptibility Diuretics Enzymes Essential Hypertension Exons Exposure to Generations Genes Guide RNA Heart failure Human Hypertension Isoenzymes Leucine Zippers Life Life Cycle Stages Mammals Measures Mediating Methods Modeling Molecular Morbidity - disease rate Mus Muscle Muscle relaxation phase Myosin ATPase Outcome Pathogenesis Pathway interactions Peripheral Resistance Phosphotransferases Prevention Process Protein Isoforms RNA Editing Reactive Oxygen Species Reading Frames Resistance Risk Factors Role Signal Pathway Signal Transduction Smooth Muscle Smooth Muscle Myocytes Smooth Muscle Myosins Somatic Cell Stress Stress Tests Stroke Testing Therapeutic Time Translating Translations Vascular Smooth Muscle Vascular resistance Vasoconstrictor Agents Vasodilation Vasodilator Agents Viral Weaning blood pressure control blood pressure reduction early life stress experimental study genome editing hypertension prevention hypertensive in vivo innovation insight lifetime risk maternal separation mortality myosin phosphatase novel novel strategies novel therapeutics oxidation stressor success

项目摘要

Project Summary High blood pressure is endemic, and despite vasodilator and diuretic therapy still accounts for much world-wide cardiovascular morbidity (heart failure, stroke) and mortality. Our studies focus on Myosin Phosphatase (MP) which by de-phosphorylating myosin causes smooth muscle relaxation. MP is the target of constrictor and dilator signaling pathways that regulate vascular tone and thereby control BP. We have proposed a model in which alternative splicing of Exon 24 (E24) of the MP regulatory subunit Mypt1 tunes vascular smooth muscle sensitivity to NO/cGMP-mediated vasorelaxation. Inclusion of the 31 nt E24 shifts the reading frame, thus coding for a C- terminal sequence lacking the leucine zipper (LZ) motif required for cGMP-dependent kinase (cGK1α) activation of MP and vasorelaxation. This vasodilator pathway may also be activated by ROS mediated oxidation of cGK1α and its downstream targets including MP. The increased vascular resistance of hypertension may in part be due to reduced bio-availability of NO and increased ROS generation reducing vasodilator signaling and increasing vascular resistance. Here we propose to test the hypothesis that precision editing of Mypt1 E24 will reduce vascular resistance to blood flow and permanently lower blood pressure (BP) in hypertension. Aim 1 uses complementary approaches of A) Cre-Lox B) Adeno-Associated Viral delivery of Crispr/Cas9, for precision editing of E24 to test if this approach can reverse vasodysfunction in the AngII model of hypertension. It compares approaches of primordial prevention vs treatment after hypertension is established. Molecular assays will determine how NO/cGMP/ROS activate MP, testing a novel 2-pool “brake and accelerator” model for the integration of dilator and constrictor signals in the control of MP, and thus blood pressure, in hypertension. The hypertensive diathesis may initiate early in life as evidenced by tracking of BP throughout the life course. Lifetime BP is most strongly related to cardiovascular outcomes, and effective lowering of BP in maturity does not normalize cardiovascular morbidity and mortality. These provide compelling rationale for the study of programming of hypertension and its primordial prevention early in life. We have shown that the switch to the E24+ (“cGMP resistant”) isoform of Mypt1 occurs during adolescence as part of arterial maturation and concordant with increasing vasoconstrictor function and BP. This process is accelerated by early life stress, an important risk factor in the development of hypertension. Aim 2 will test the ability of precision editing of E24 early in life to mitigate the deleterious effect of stress early, or throughout, the life course, on arterial function and programming. It will also test if primordial prevention of vasodysfunction via precision editing of E24 in early life is more effective as compared to after hypertension is well established. We expect that this novel strategy of precision editing of the Mypt1 alternative exon 24, by shifting the MP isozyme pools to favor vasodilator signaling, will cause vasodilator sensitization and lower BP throughout the life course. If successful this experimental strategy has high potential for translation as a new therapeutic in humans.

项目概要高血压是一种地方病，尽管血管扩张剂和利尿剂治疗在世界范围内仍然占很大比例我们的研究重点是肌球蛋白磷酸酶 (MP)。通过去磷酸化肌球蛋白导致平滑肌松弛，MP 是收缩肌和扩张肌的目标。我们提出了一个模型，其中调节血管张力和控制血压。 MP调节亚基Mypt1的外显子24（E24）的选择性剪接调节血管平滑肌敏感性 NO/cGMP 介导的血管舒张作用，包含 31 nt E24 会改变阅读框，从而编码 C-。末端序列缺少 cGMP 依赖性激酶 (cGK1α) 激活所需的亮氨酸拉链 (LZ) 基序 MP 和血管舒张作用也可能被 ROS 介导的 cGK1α 氧化激活。及其下游目标（包括 MP）高血压的血管阻力增加可能部分是由于。降低 NO 的生物利用度并增加 ROS 的产生，减少血管舒张信号传导并增加在这里，我们建议检验 Mypt1 E24 的精确编辑会降低的假设。血管对血流的阻力并永久降低高血压患者的血压 (BP)。使用 A) Cre-Lox B) Crispr/Cas9 腺相关病毒传递的补充方法，以实现精确性编辑 E24 以测试这种方法是否可以逆转高血压 AngII 模型中的血管功能障碍。比较建立高血压后的原始预防和治疗方法。将确定 NO/cGMP/ROS 如何激活 MP，测试新型 2 池“制动器和加速器”模型扩张器和收缩器信号在 MP 控制中的整合，从而在高血压中控制血压。一生中血压追踪可证明高血压素质可能在生命早期就开始终生血压与心血管结局以及成熟期血压的有效降低密切相关。并没有使心血管发病率和死亡率正常化，这些为研究提供了令人信服的理由。高血压的规划及其生命早期的初步预防我们已经表明，转向高血压。 Mypt1 的 E24+（“cGMP 抗性”）亚型出现在青春期，是动脉成熟的一部分，并且与血管收缩功能和血压的增加相一致，早期生活压力会加速这一过程。 Aim 2将测试E24的精准编辑能力。在生命早期减轻压力对动脉功能和整个生命过程的有害影响它还将测试是否可以在生命早期通过精确编辑 E24 来预防血管功能障碍。我们预计这种新策略比高血压确立后更有效。通过改变 MP 同工酶池以支持血管舒张剂，精确编辑 Mypt1 替代外显子 24 如果成功的话，将导致血管扩张剂敏化并降低整个生命过程中的血压。实验策略具有作为人类新疗法的巨大潜力。